目的 基于二維液相色譜-高分辨串聯(lián)質(zhì)譜（2D-LC-HR- MS/MS）法分析頭孢丙烯原料藥雜質(zhì)譜。方法 一維色譜條件進(jìn)行樣品色譜圖采集，確認(rèn)各雜質(zhì)的出峰位置，采用 YMC Hydrosphere 色譜柱（250 mm×4.6 mm，5 μm），以 11.5 g·L^-1磷酸二氫銨（用磷酸調(diào)節(jié) pH 為 4.4）為流動(dòng)相 A，乙腈-流動(dòng)相 A（50∶50）為流動(dòng)項(xiàng) B，梯度洗脫；柱溫 40 ℃；體積流量1.0 mL·min-1；檢測(cè)波長(zhǎng) 230 nm；進(jìn)樣量 4 μL。二維液相脫鹽后切進(jìn)高分辨串聯(lián)質(zhì)譜進(jìn)行分析，根據(jù)結(jié)果推斷雜質(zhì)結(jié)構(gòu)及生成機(jī)制，采用 Waters BEH C₁₈色譜柱（50 mm×2.1 mm，1.7 μm），以 0.01% 甲酸水溶液為流動(dòng)相 A，乙腈為流動(dòng)相 B，切峰后開始 A 相由 98% 到 1%，柱溫 40 ℃，體積流量 0.3 mL·min-1，質(zhì)譜采用 Xevo G2-XS QTof MS 系統(tǒng)，離子源為 ESI 源，毛細(xì)管電壓 3.0 kV，霧化器溫度 450 ℃，掃描范圍 m/z 100～2 000。結(jié)果 頭孢丙烯樣品中存在 9 個(gè)雜質(zhì)色譜峰，其中 5 個(gè)雜質(zhì)為已知雜質(zhì)，峰 3 為雜質(zhì) B（頭孢羥氨芐）、峰 5 為雜質(zhì) D、峰 6 為雜質(zhì) F、峰 7 為雜質(zhì) G、峰 9 為雜質(zhì) I；對(duì)其中 3 個(gè)未知雜質(zhì)可能的結(jié)構(gòu)式進(jìn)行了初步推測(cè)以及探討了可能的生成途徑，峰 2 分子式為 C₁₈H₁₉N₃O₆S，該化合物比頭孢丙烯多一個(gè)氧，分析其為頭孢丙烯的氧化雜質(zhì)；峰 4 分子式為 C₁₆H₁₅N₃O₆S，與雜質(zhì) B 相比增加了 1 個(gè)氧原子，減少了 2 個(gè)氫原子，判斷其為 7-ACA 內(nèi)酯與對(duì)羥基苯甲甘氨酸甲酯（HPGM）反應(yīng)產(chǎn)物中的硫原子繼續(xù)發(fā)生了氧化生成的；峰 8 分子式為 C₈H₉NO₂S，該組分為頭孢丙烯分子結(jié)構(gòu)的一部分。峰 1 有待進(jìn)一步研究。結(jié)論 該方法有效解決了頭孢丙烯流動(dòng)相中含不揮發(fā)性磷酸鹽的色譜體系與色譜-質(zhì)譜快速鑒定雜質(zhì)不兼容的難題，可以簡(jiǎn)單、快速地對(duì)頭孢丙烯有關(guān)物質(zhì)進(jìn)行定性分析及雜質(zhì)譜研究。

Objective To analyze the impurity profile of cefprozil API using 2D-LC-HR-MS/MS. Methods The sample chromatogram was acquired by one-dimensional chromatography conditions to confirm the peak positions of each impurity. The YMC Hydrosphere column (250 mm×4.6 mm, 5 μm) was used, with 11.5 g·L^-1 ammonium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid) as solvent A, a mixture of acetonitrile and solvent A (50:50) as solvent B, and gradient elution; column temperature was 40℃; flow rate was 1.0 mL·min-1; detection wavelength was 230 nm; injection volume was 4 μL. The impurity profile was analyzed by 2D-LC desalting and high-resolution tandem mass spectrometry (HR-MS/MS) after cutting into the mass spectrometer. Based on the results, the structures and formation mechanisms of the impurities were inferred. The Waters BEH C₁₈ column (50 mm×2.1 mm, 1.7 μm) was used, with 0.01% formic acid aqueous solution as solvent A and acetonitrile as solvent B. The peaks were cut and the A phase was gradually increased from 98% to 1% starting from the cut. The column temperature was 40° C, the flow rate was 0.3 mL·min-1, and the mass spectrometer used was the Waters Xevo G2-XS QTof MS system with an ESI ion source, a capillary voltage of 3 kV, a vaporizer temperature of 450 ℃, and a scanning range of m/z 100—2 000. Results Nine impurity peaks were identified in the cefprozil sample, of which five were known impurities. Peak 3 was impurity B (cefhydroxime), peak 5 was impurity D, peak 6 was impurity F, peak 7 was impurity G, and peak 9 was impurity I. Preliminary structural hypotheses were made for the three unknown impurities and the possible generation routes were discussed. Peak 2 has a molecular formula of C₁₈H₁₉N₃O₆S, which was one oxygen atom more than cefprozil. It was analyzed as an oxidation impurity of cefprozil. Peak 4 has a molecular formula of C₁₆H₁₅N₃O₆S, which was one oxygen atom and two hydrogen atoms less than impurity B. It was judged that it was the sulfur atom of the reaction product between 7-ACA lactam and HPGM (hydroxyphenylglycine methyl ester) continuing to undergo oxidation. Peak 8 has a molecular formula of C₈H₉NO₂S, which is part of the molecular structure of cefprozil. Peak 1 needs further study. Conclusion This method effectively solved the problem of the chromatographic system with non-volatile phosphate in the mobile phase of cefprozil flowing phase and the incompatibility of chromatography-mass spectrometry rapid identification of impurities. It can simply and quickly determine the qualitative analysis and impurity spectrum of cefprozil-related substances.

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廣東省重點(diǎn)領(lǐng)域研發(fā)計(jì)劃項(xiàng)目（2022B1111070004）；國(guó)家自然科學(xué)基金重點(diǎn)項(xiàng)目（61633006）